Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube idealized folding reaction so intensively studied over the past several decades, that is crucial to the fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to arise from defects in protein folding, either because of the loss or alteration of essential protein functions, or because of the build-up of toxic species such as aggregates. We believe that novel methods and creative collaborations will allow us to overcome the daunting technical obstacles that have impeded progress on protein folding in the cell. Focusing first on a small model protein for which we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging methods will be used to insure that observed signals report on relevant phenomena and to reveal novel functionally significant spatial localization patterns. We anticipate that this research will lead to new paradigms for how amino acid sequences encode folding information and that the resulting enhanced understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and aggregation diseases.